In eukaryotes, the family of copper and zinc containing superoxide dismutases (SOD1) are known to participate in anti-oxidant defense and cell signaling. Very recently, the Culotta laboratory has uncovered a new class of SOD1-like molecules in eukaryotes that function without a zinc ion. The prototype of this family is Candida albicans SOD5, an extracellular copper-only SOD that is essential for virulence of the fungal pathogen. Unlike SOD1, SOD5 has no zinc site and contains an unusually open copper site due to absence of an electrostatic loop VII. When secreted from C. albicans, SOD5 can rapidly acquire its copper co-factor from extracellular pools of the metal. These unique features in metal co-factors displayed by SOD5 vs the canonical SOD1 may represent adaptations to host-mediated changes in copper and zinc during infection. To begin to understand the novel metallobiology of C. albicans SOD5, we shall use a combination of biochemical, spectroscopic and cell biology approaches to explore mechanisms by which SOD5 operates without zinc and how the enzyme is charged with copper during infection. Aim 1: To determine how SOD5 functions without a zinc metal ion cofactor: Analysis of the three dimensional structure of Cu-SOD5 has revealed a hydrogen bond network to the copper site that may substitute for zinc in this enzyme. This network involves conserved residues E110 and D113 that in preliminary studies have been shown to be important for maximal SOD5 activity. Using a Pichia pastoris yeast expression system for secretory proteins, we will express and purify large quantities of extracellular SOD5 E110 and D113 mutants. We will characterize their respective metal binding capabilities and obtain kinetic measurements of catalysis using pulse radiolysis. These studies will reveal whether the role of E110 and D113 in Cu-SOD5 catalysis is analogous to the role of zinc in SOD1. Aim 2. To understand the role of host copper in the activation of SOD5 for pathogen defense: C. albicans relies on its animal host for acquiring copper and one intriguing source is the copper burst of macrophages - a defense strategy to kill pathogens through copper toxicity. Since SOD5 is rapidly charged with extracellular copper, it may take advantage of the copper burst to charge itself for anti-oxidant defense. By binding excess copper, SOD5 might also help protect C. albicans from host-mediated copper toxicity. To address this, we will test whether extracellular SOD5 has the capacity to protect C. albicans from copper toxicity in yeast cultures and in macrophage infection systems. By creating copper deficient macrophages, we will test whether SOD5 secreted from C. albicans is charged with copper from the macrophage, and whether this pool of macrophage copper is important for pathogen killing during infection. Together, these studies will increase our basic understanding of Candida albicans SOD5 at both the biochemical and cellular levels and may ultimately lead to the development of new therapies for candidiasis directed at the novel copper-only SODs of C. albicans.